ES2784830T3 - Methods for the diagnosis and treatment of autoimmune disease as a consequence of multiple sclerosis - Google Patents

Abstract

A method of selecting a multiple sclerosis (MS) patient for lymphocyte depletion therapy, comprising the steps of: a) measuring the level of IL-21 in a blood sample from the patient, wherein the blood sample is obtained prior to any lymphocyte depletion therapy, b) comparing the level of IL-21 with the level of IL-21 in a control blood sample from a subject without an autoimmune disease, and c) selecting the patient for therapy lymphocyte depletion if the level of IL-21 in the patient's blood sample is not elevated compared to the level of IL-21 in the control blood sample.

Description

DESCRIPTION

Methods for the diagnosis and treatment of autoimmune disease as a consequence of multiple sclerosis

BACKGROUND OF THE INVENTION

Multiple sclerosis ("MS") is an inflammatory autoimmune disorder of the central nervous system (Compston and Coles, Lancet 372, 1502-17 (2008)). With a prevalence of around one in 1000, MS is the most common cause of neurological disability in young adults (Polman and Uitdehaag, BMJ 321, 490-4 (2000)). MS involves the involvement of the immune system, acute inflammatory injury to axons and glia, recovery of function and structural repair, post-inflammatory gliosis and neurodegeneration (see, for example, Compston and Coles, 2008) . These sequential processes underlie a clinical course characterized by recovery episodes, episodes that leave persistent deficits, and secondary progression. Idem.

The goal of MS treatment is to reduce the frequency and severity of relapses, prevent disability resulting from disease progression, and promote tissue repair (Compston and Coles, 2008). The main approach to treating MS is the modulation or suppression of the immune system. Currently available MS drugs include interferon beta-Ia (eg AVONEX and REBIF), interferon beta-Ib (eg BETASERON), glatiramer acetate (eg COPAXONE), mitoxantrone (eg NOVANTRONE), and natalizumab (eg, TYSABRI). Another promising new drug for MS is alemtuzumab (CAMPATH-1H).

Alemtuzumab is a humanized monoclonal antibody directed against CD52, a protein widely distributed on the surface of lymphocytes and monocytes but with an unknown function. Alemtuzumab has been used to treat B-cell chronic lymphocytic leukemia. A single pulse of treatment leads to rapid, profound, and prolonged lymphopenia. Cell numbers recover but at variable rates; CD4 + T cells are particularly slow to recover, remaining depleted for at least five years (Coles et al., Journal of Neurology 253, 98-108 (2006)). A phase 2 trial (CAMMS-223 study group; Coles et al., N. Engl. J. Med. 359, 1786-1801 (2008)) has shown that alemtuzumab is highly effective in the early treatment of multiple sclerosis. recurring-sender. This drug reduces the risk of disease activity and accumulation of disability by more than 70% compared to interferon beta in patients with early-remitting relapsing multiple sclerosis. The main adverse effect is autoimmunity, which arises in the context of T-cell lymphopenia months or years after administration. About 20-30% of patients develop thyroid autoimmunity, primarily Graves disease (Coles et al., Lancet 354, 1691-1695 (1999)), and 3% have immune thrombocytopenia (ITP) (Coles et al. ., 2008). Single cases of Goodpasture's disease, autoimmune neutropenia (Coles et al., Journal of Neurology 253, 98-108 (2006)), and autoimmune hemolytic anemia (unpublished observation) have also been observed. Furthermore, another 5.5% of patients develop sustained non-thyroid autoantibodies without clinical disease (Coles et al., 2006). The timing and spectrum of autoimmunity after alemtuzumab is similar to that seen in other examples of "reconstitution autoimmunity" in other clinical settings; For example, autoimmune thyroid disease and autoimmune cytopenias also predominate months or years after hematopoietic stem cell transplantation or HIV antiretroviral therapy (Chen et al., Medicine (Baltimore) 84, 98-106 (2005); Daikeler and Tyndall, Best. Pract. Res. Clin. Haematol. 20, 349-360 (2007); Jubault et al., J. Clin. Endocrinol. Metab. 85, 4254-4257 (2000); Ting, Ziegler, and Vowels, Bone Marrow Transplant. 21, 841-843 (1998); Zandman-Goddard and Shoenfeld, Autoimmun. Rev. 1,329-337 (2002)).

Although the autoimmunity that arises in the context of lymphopenia is well recognized in animal models, it is rarely found and thus difficult to study in humans. Most lymphopenic subjects do not develop autoimmunity, suggesting that additional factors are involved (Krupica et al., Clin Immunol 120, 121-128 (2006)). It is not clear what those additional factors are. Krupica et al. (Clinical Immunology 20: 1, 121-128 (2006)) refer to IL-21 overproduction as a potential factor to induce autoimmunity during lymphopenia in NOD mice. The depletion of regulatory T cells has been considered as a factor, as seen in murine colitis and gastritis models (Alderuccio et al., J Exp. Med 178, 419-426 (1993); McHugh et al., J Immunol 168, 5979-5983 (2002); Powrie et al., Int. Immunol 5, 1461-1471 (1993); Sakaguchi et al., J Immunol 155, 1151-1164 (1995)). However, regulatory T cells have been observed to increase after alemtuzumab in human patients and subsequently return to normal levels (Cox et al., Eur J Immunol 35, 3332-3342 (2005)). This observation has since been replicated (Bloom et al., Am J Transplant. 8, 793-802 (2008)), and is consistent with other experimental lymphopenic models (de Kleer, I. et al., Blood 107, 1696 1702 (2006); Zhang, H. et al., Nat Med 11, 1238-1243 (2005)).

SUMMARY OF THE INVENTION

We have invented new and useful methods and compositions to improve risk management in the treatment of MS. The methods and compositions reduce the side effects of MS treatment, such as secondary autoimmunity, and help healthcare providers and patients select regimens for the treatment of MS and post-treatment monitoring. The methods of this invention are based on our discovery that in patients with multiple sclerosis (MS), elevated IL-21, detectable even before lymphocyte depletion therapy, such as alemtuzumab therapy, is correlated with increased risk of developing secondary autoimmunity after therapy. Furthermore, we have discovered that an individual's IL-21 level can be genetically determined: the genotypes of single nucleotide polymorphisms (SNPs) of A / A in SNP rs13151961, G / G in SNP rs6822844, and C / C in SNP rs6840978, are associated with elevated IL-21.

The invention is defined according to the claims. Thus, the invention provides a method of selecting a multiple sclerosis (MS) patient for lymphocyte depletion therapy, comprising the steps of:

a) measuring the level of IL-21 in a blood sample from the patient, wherein the blood sample is obtained prior to any lymphocyte depletion therapy;

b) comparing the level of IL-21 with the level of IL-21 in a control blood sample from a subject without an autoimmune disease, and

c) selecting the patient for lymphocyte depletion therapy if the level of IL-21 in the patient's blood sample is not elevated compared to the level of IL-21 in the control blood sample. In one embodiment, the measurement is of mRNA encoding IL-21 in IL-21 producing cells in the sample. In one embodiment, the IL-21 producing cells are Th17 cells. In one embodiment, the measurement is intracellular IL-21. In one embodiment, the measurement comprises staining and flow cytometry. In one embodiment, the measure is serum IL-21. In one embodiment, the measurement comprises the use of an enzyme-linked immunosorbent assay (ELISA). In one embodiment, lymphocyte depletion therapy comprises administering an anti-CD52 antibody to the patient. In one embodiment, the anti-CD52 antibody is alemtuzumab.

The invention also provides a method of selecting a multiple sclerosis (MS) patient for lymphocyte depletion therapy, comprising the steps of:

a) genotype a patient DNA sample to detect the presence or absence of one or more genotypes of single nucleotide polymorphisms (SNPs) selected from the group consisting of: A / A in SNP rs13151961, G / G in SNP rs6822844, and ^c / ^c in SNP rs6840978, in which the DNA sample is obtained prior to any lymphocyte depletion therapy, and

b) selecting the patient for lymphocyte depletion therapy if said genotypes of SNPs are not detected in the DNA sample.

In one embodiment, lymphocyte depletion therapy comprises administering an anti-CD52 antibody to the patient. In one embodiment, the anti-CD52 antibody is alemtuzumab.

Here we describe methods to identify an MS patient who has an elevated interleukin-21 (IL-21) compared to IL-21 in a subject without an autoimmune disease. The methods may comprise the step of measuring IL-21 in a blood sample from the MS patient, thus identifying an MS patient having elevated IL-21 compared to said subject. Alternatively, the methods may comprise the step of genotyping the patient to detect the presence or absence in the patient of one or more genotypes of single nucleotide polymorphisms (SNPs) associated with elevated IL-21, such as those selected from the group consisting in: A / A in SNP rs13151961, G / G in SNP rs6822844, and C / C in SNP rs6840978, in which the presence of one or more of said genotypes is associated with elevated IL-21.

Also described herein are methods of identifying an MS patient who is at increased risk of developing a secondary autoimmune disease after lymphocyte depletion. The methods may comprise the step of determining (for example, measuring) the level of interleukin-21 (IL-21) in a blood sample from the MS patient, in which an elevated level of IL-21 compared to a subject without an autoimmune disease indicates that the patient has a higher risk of developing a secondary autoimmune disease compared to MS patients without elevated IL-21. Alternatively, the methods may comprise the step of determining (e.g., by genotyping) the presence or absence in the patient of one or more genotypes of single nucleotide polymorphisms (SNPs) associated with elevated IL-21, such as those selected from the group consisting of: A / A in SNP rs13151961, G / G in SNP rs6822844, and C / C in SNP rs6840978, in which the presence of one or more (for example, two or three) of said genotypes is associated with an increased risk of developing a secondary autoimmune disease compared to MS patients without said one or more genotypes. Optionally, these methods comprise the step of informing the patient and / or their healthcare provider about said increased risk, and / or the step of recording the increased risk.

Also described herein are methods of selecting or identifying a patient with MS in need of further monitoring for the development of a secondary autoimmune disease after lymphocyte depletion therapy. These methods may comprise the step of measuring IL-21 in a blood sample of the MS patient, wherein the elevated IL-21 in said patient compared to a subject without an autoimmune disease indicates that the patient needs further monitoring to the development of a secondary autoimmune disease compared to MS patients without elevated IL-21. Alternatively, the methods may comprise the step of genotyping the patient to detect the presence or absence of one or more genotypes of SNPs associated with elevated IL-21, such as those selected from the group consisting of: A / A in SNP rs13151961, G / G in SNP rs6822844, and C / C in SNP rs6840978, in which the presence of one or more of said SNPs indicates that the patient needs greater monitoring for the development of a secondary autoimmune disease compared to MS patients without said one or more genotypes. Optionally, these methods comprise the step of informing the patient and / or their healthcare provider of the need for further monitoring and / or the step of recording the need.

The invention also provides methods for reporting a treatment for a patient with MS, comprising measuring IL-21 in a blood sample from said patient or genotyping the patient for the presence or absence of the three SNP phenotypes mentioned above, and selecting an appropriate treatment regimen for IL-21 measurement or genotype.

The description provides methods for treating MS in a patient known to be in need, comprising the steps of (a) obtaining or determining information about (i) IL-21 in a blood sample from the patient (e.g., measuring IL -21 in the sample); or (ii) the presence or absence of one or more genotypes of single nucleotide polymorphisms (SNPs) associated with elevated IL-21, such as those selected from the group consisting of: A / A in SNP rs13151961, G / G in SNP rs6822844 G / G, and C / C in SNP rs6840978 (eg, genotyping the patient); (b) administering a therapeutic agent for multiple sclerosis to said patient, and (c) optionally monitoring the patient for the development of a secondary autoimmune disease. The treatment methods can be used in patients who have normal levels of IL-21 and / or do not have any of the three genotypes of IL-21 SNPs mentioned above. Also described are anti-CD52 antibodies (eg, Alemtuzumab or a biologically similar agent), or antigen-binding portions thereof, which can be used in the treatment methods of the disclosure, and uses of these antibodies or binding portions. to antigen in the manufacture of a drug for use in the treatment methods of the disclosure. Therapeutic regimens using the description treatment methods are also described.

The description provides methods for reducing the occurrence or severity of a secondary autoimmune disease in a multiple sclerosis patient who has been or will be treated with lymphocyte depletion therapy, wherein the secondary autoimmune disease occurs after treatment with the therapy. of lymphocyte depletion, comprising the step of administering an IL-21 antagonist, for example before, during or after treatment with lymphocyte depletion therapy. Also described are IL-21 antagonists for use in these methods (for example, an anti-IL-21 or anti-IL-21 receptor antibody, or an antigen-binding portion thereof; or a soluble IL-21 receptor 21), and uses of these IL-21 antagonists in the manufacture of a drug for use in the methods.

The description provides methods for evaluating the responsiveness of T cells to treatment with a lymphocyte depletion therapy in a patient with multiple sclerosis, comprising measuring caspase-3 in T cells obtained from said patient after said therapy, wherein an increase in caspase-3 in said T cells compared to T cells from an MS patient not receiving said therapy is indicative of the responsiveness of the T cells to said therapy. The measurement may involve determining the amount or concentration of caspase-3 or nucleic acid that encodes caspase-3.

The description provides methods for informing a patient with MS of an increased risk of developing a secondary autoimmune disease after lymphocyte depletion, comprising the steps to obtain or determine information on interleukin-21 (IL-21) in a sample of blood from the patient with MS, wherein elevated IL-21 compared to a subject without an autoimmune disease indicates that the patient has an increased risk of developing a secondary autoimmune disease compared to MS patients without elevated IL-21; and inform the patient of an increased risk or lack thereof. Alternatively, the methods comprise obtaining or determining information on the presence or absence of one or more of the IL-21 genotypes mentioned above, rather than information on the level of IL-21 in blood. Accordingly, the disclosure also provides methods for informing an MS patient of a need, or lack of a need, for further monitoring for the development of a secondary autoimmune disease after lymphocyte depletion therapy based on the level of IL-21 from the patient or the presence or absence of the IL-21 genotypes described above.

The description provides methods for reporting a regimen for monitoring a patient with MS after lymphocyte depletion therapy, comprising the steps of obtaining or determining information on (i) IL-21 in a blood sample from the patient; or (ii) the presence or absence of one or more genotypes of single nucleotide polymorphisms (SNPs) associated with elevated IL-21, such as those selected from the group consisting of: A / A in SNP rs13151961, G / G in SNP rs6822844 G / G, and C / C in SNP rs6840978; and selecting an appropriate monitoring regimen for the patient based on the information. An appropriate monitoring regimen may include, for example, the measurement of autoantibodies in the patient.

The present invention provides advantages in risk management in the treatment of MS. For example, the disclosure provides methods for delivering a lymphocyte depleting drug to a patient to treat multiple sclerosis, comprising the steps of advising the patient about the increased risk of developing a secondary autoimmune disease after treatment with said drug, in the one with the highest risk associated with (i) elevated IL-21; or (ii) the presence of one or more genotypes of single nucleotide polymorphisms (SNPs) associated with elevated IL-21, such as those selected from the group consisting of: A / A in SNP rs13151961, G / G in SNP rs6822844 G / G, and C / C in SNP rs6840978; and providing the drug to the patient after such counseling, optionally after obtaining the patient's written authorization.

The disclosure further provides methods for identifying an individual who is likely to have elevated levels of interleukin-21 (IL-21) compared to a subject without any known inflammatory condition, comprising the step of genotyping the individual to detect the presence or absence. of one or more genotypes of single nucleotide polymorphisms (SNPs) associated with elevated IL-21, such as those selected from the group consisting of: A / A in SNP rs13151961, G / G in SNP rs6822844, and C / C in SNP rs6840978, in which the presence of one or more of said genotypes is associated with elevated IL-21.

In the context of this invention, lymphocyte depletion can be induced by a treatment that targets CD52, for example a treatment with an anti-CD52 antibody (eg, a monoclonal antibody) or an antigen-binding portion thereof. . The anti-CD52 antibody can be alemtuzumab or a biologically similar agent, such as an antibody that competes to bind CD52 with alemtuzumab.

In the methods of this invention, the measurement of IL-21 may involve measuring (e.g., detecting / quantifying) the amount or concentration of IL-21 or nucleic acid encoding IL-21 in a sample, or the amount or concentration of MRNA encoding IL-21 in IL-21 producing cells (eg, Th17 cells) in the sample. In some embodiments, the measurement is intracellular IL-21, using, for example, cytokine staining and flow cytometry. In some embodiments, the measurement is serum IL-21, using, for example, an enzyme-linked immunosorbent assay (ELISA). The description encompasses ELISA kits for detecting IL-21 levels in a human subject, comprising an anti-IL-21 antibody, or an antigen-binding portion thereof, or a soluble IL-21 receptor. The kits may further include an instruction instructing the user to take a blood sample from a human subject.

IL-21 information (including measurement or genotyping) can be obtained before, during, or after MS therapy. The methods of this invention can be used in the context of any form of MS, including, but not limited to, relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis.

The disclosure also provides kits for treating multiple sclerosis, comprising a lymphocyte depleting therapeutic agent (eg, an anti-CD52 antibody such as alemtuzumab); and a written instruction to inform a patient or healthcare provider of the potential for an increased risk of developing a secondary autoimmune disease after treatment with said agent, wherein the increased risk is indicated by or associated with (i) Elevated IL-21, or (ii) the presence of one or more genotypes of single nucleotide polymorphisms (SNPs) associated with elevated IL-21, such as those selected from the group consisting of: A / A in SNP rs13151961, G / G in SNP rs6822844 G / G, and C / C in SNP rs6840978.

The disclosure further provides kits for identifying an MS patient who is at increased risk of developing a secondary autoimmune disease after lymphocyte depletion, comprising an anti-interleukin-21 (IL-21) antibody and one or more reagents to detect the binding of said antibody to IL-21 in a blood sample of the patient with MS. The description also provides kits for identifying an MS patient who is at increased risk of developing a secondary autoimmune disease after lymphocyte depletion, comprising one or more reagents suitable for identifying the genotype of one or more single nucleotide polymorphisms (SNPs ) selected from the group consisting of: SNP rs13151961, SNP rs6822844, and SNP rs6840978, in a sample obtained from an individual.

Other features and advantages of the invention will be apparent from the following figures and detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a graph showing T-cell precursor frequency (PF) from healthy controls (HC), untreated patients (Pre), and at 3-month intervals after alemtuzumab, unstimulated (Unstim), or after basic protein culture myelin receptor (MBP) or thyroid stimulating hormone receptor (TSHr). (* p <0.05, ** p <0.01, *** p <0.001)

FIG. 1B is a graph showing the proliferative index (PI) of T cells from healthy controls (HC), untreated patients (Pre) and at 3-month intervals after alemtuzumab, unstimulated (Unstim), or after basic protein culture myelin receptor (MBP) or thyroid stimulating hormone receptor (TSHr). (* p <0.05, ** p <0.01, *** p <0.001)

FIG. 1C is a graph showing the total number of viable T cells after 10 days in culture of healthy controls (HC), untreated patients (Pre), and at 3-month intervals after alemtuzumab, unstimulated (Unstim), or after culture with myelin basic protein (MBP) or thyroid stimulating hormone receptor (TSHr). (* p <0.05, ** p <0.01, *** p <0.001)

FIG. 1D is a graph showing the percentage of T cells that undergo apoptosis in response to no stimulus or after culturing with MBP or TSHr in culture of healthy controls (HC), untreated patients (Pre), and at 3-month intervals after alemtuzumab . (* p <0.05, ** p <0.01, *** p <0.001)

FIG. 1E are graphs and a graph showing passive T-cell apoptosis of healthy controls and patients before and after alemtuzumab at 3-month intervals. (* p <0.05, ** p <0.01, *** p <0.001) FIG. 1F are graphs and a graph showing Fas-mediated T-cell apoptosis of healthy controls and patients before and after alemtuzumab at 3-month intervals. (* p <0.05, ** p <0.01, *** p <0.001)

FIG. 1G is a graph showing CD4 + and CD8 + T cell passive apoptosis of healthy controls, pretreatment patients, and 9 months after alemtuzumab. (* p <0.05, ** p <0.01, *** p <0.001)

FIG. 1H is a graph showing Fas-mediated CD4 + and CD8 + T cell apoptosis of healthy controls, pretreated patients, and at 9 months after alemtuzumab. (* p <0.05, ** p <0.01, *** p <0.001) FIGS. 2A-2C are graphs showing caspase 3 mRNA expression relative to beta-actin mRNA expression in (A) CD3 + T cells, (B) CD14 + monocytes, and (C) CD19 + B cells, respectively, immediately ex vivo or after stimulation with MBP or polyclonal stimulation (anti-CD3 / 28 antibodies). (* p <0.05, ** p <0.01, *** p <0.001)

FIG. 3 are graphs and a graph showing that autoimmunity after alemtuzumab is associated with excessive T-cell apoptosis. Separate graphs show the percentage of T-cell apoptosis that is passive (Un), Fas-mediated, or in response to stimulation of MBP or r TSH in those without autoimmunity (Ge et al., Proceedings of the National Academy of Sciences of the United States of America 101, 3041-3046 (2004)) or those with secondary autoimmunity (Ge et al., 2004). (* p <0.05, ** p <0.01, *** p <0.001)

FIGS. 4A and 4B are graphs showing that rhIL-21 induces T cell apoptosis in vitro. They show that (A) CD4 + T cells and (B) CD8 + T cells, respectively, unstimulated or polyclonally stimulated (anti-CD3 / CD28), undergo apoptosis in response to rhIL-21 in a dose-dependent manner. (* p <0.05, ** p <0.01, *** p <0.001)

FIGS. 5A-5D are graphs showing that rhIL-21 induces T cell proliferation in vitro. FIG.

5A is a graph showing the proliferative rate of unstimulated CD4 + and CD8 + T cells in response to rhIL-21. FIG. 5B is a graph showing the proliferative rate of polyclonally stimulated CD4 + and CD8 + T cells (anti-CD3 / CD28) in response to rhIL-21. FIG. 5C is a graph showing the precursor frequency of unstimulated CD4 + and CD8 + T cells in response to rhIL-21. FIG. 5D is a graph showing the precursor frequency of polyclonally stimulated CD4 + and CD8 + T cells in response to rhIL-21. (* p <0.05, ** p <0.01, *** p <0.001)

FIG. 5E are graphs showing the number of unstimulated or polyclonally stimulated CD4 + or CD8 + cells (anti-CD3 / CD28) in different channels in the absence of, or in response to, rhIL-21.

FIG. 6A is a graph showing serum IL-21 before and after alemtuzumab treatment in 15 patients with and 15 patients without secondary autoimmunity. (* p <0.05, ** p <0.01, *** p <0.001)

FIG. 6B is a graph showing pre-treatment serum IL-21 levels (pg / ml) in non-autoimmune patients (those who did not have post-alemtuzumab autoimmunity) and autoimmune patients (those who had post-alemtuzumab autoimmunity) .

DETAILED DESCRIPTION

This invention is based on our discovery that the occurrence of secondary autoimmunity in an MS patient after lymphocyte depletion therapy (for example, after treatment with alemtuzumab) is associated with elevated IL-21 in the patient. We have found that IL-21 is elevated compared to norm (see explanations below) even before therapy in MS patients who then develop secondary autoimmunity after therapy. We have also found that after lymphocyte depletion therapy, IL-21 rises even more dramatically in those same patients, compared to MS patients without signs of secondary autoimmunity, whose IL-21 rises to a very high degree. less. Thus, IL-21 levels predict the onset of secondary autoimmunity after lymphocyte depletion therapy in a patient with MS. We have also discovered that the genotypes of single nucleotide polymorphisms (SNPs) of A / A in SNP rs13151961, G / G in SNP rs6822844, and C / C in SNP rs6840978 are associated with elevated IL-21 in an individual; thus, genotyping a patient with MS for the presence or absence of these specific SNP genotypes also helps to predict the risk of developing secondary autoimmunity in the patient after lymphocyte depletion.

We first described the autoimmunity complicating treatment with alemtuzumab (CAMPATH-1H) in 1999 (Coles et al., 1999), and we have continued to look at this complication of what is increasingly recognized as a highly effective therapy for recurrent multiple sclerosis. -remitter (Coles et al., J Neurology 253, 98-108 (2006); Coles et al., 1999 and 2008). Our studies described below included a number of available patient cohorts and aimed to understand this unprecedented “pattern” of human autoimmunity that occurs in a subset of MS patients treated with alemtuzumab.

Immune status is radically altered after exposure to alemtuzumab. T cells that regenerate in the lymphopenic environment generated by alemtuzumab are highly proliferative and prone to self-reactivity. However, these cells are highly unstable and short-lived. While their fate has not been directly addressed previously (King et al., Cell 117, 265-277 (2004)), we show that these cells are rapidly dying from apoptosis. Sustained high levels of T-cell apoptosis may explain why a single dose of alemtuzumab induces T-cell lymphopenia that lasts for several years, although the circulating half-life of alemtuzumab is only six days and the hematologic precursors are not depleted (Gilleece et al., Blood 82, 807-812 (1993)).

In this context, we show that patients with secondary autoimmunity have higher rates of T-cell apoptosis, but not higher T-cell lymphopenia, than those without autoimmunity, suggesting a longer cell cycle in this group. These T cell cycle disturbances are associated with significantly higher serum IL-21 expression, which we have found to be genetically determined in at least some cases. Furthermore, susceptibility to lymphopenia-associated autoimmunity manifests before lymphocyte depletion, with pre-treatment IL-21 levels accurately predicting (positive predictive value greater than 70%, for example 83%, and negative predictive of more than 62%, for example 72%) the development of autoimmunity months to years after exposure to alemtuzumab. Without wishing to be bound by any theory, we believe that by driving T cell expansion and death cycles to excess, IL-21 increases stochastic opportunities for T cells to encounter autoantigen and break tolerance, thereby promoting autoimmunity. .

In summary, our findings provide the first exploration of lymphopenia-induced autoimmunity in man, and provide a conceptual framework for understanding lymphopenia-associated autoimmunity that goes beyond the narrow context of treating multiple sclerosis with alemtuzumab. The concept is that, first, the therapeutic depletion of lymphocytes, and second, the genetically restricted overproduction of IL-21, leads to a state of excess T-cell cycling and reduced survival, promoting autoimmunity. in humans. These findings provide a basis for the present invention.

This invention provides methods for managing MS patients when considering lymphocyte depletion therapy such as alemtuzumab therapy. For example, our invention provides methods for identifying an MS patient who has elevated IL-21 compared to norm (i.e., IL-21 levels in control subjects as described below), and methods for identifying a MS patient who is at increased risk of developing secondary autoimmunity after lymphocyte depletion. These methods comprise the step of measuring IL-21 (eg, intracellular or extracellular protein levels, RNA transcript levels, or IL-21 activity levels; see explanations below) in a patient's blood sample, and comparing the IL-21 value with the normal IL-21 value. Alternatively, instead of or in addition to blood testing, the patient can be genotyped to detect the presence or absence of one or more SNP genotypes of A / A in ^s N ^p rs13151961, G / G in SNP rs6822844, and C / C in SNP rs6840978, in which the presence of one, two or all three genotypes is associated with elevated IL-21. As discussed above, elevated IL-21 is associated with an increased risk of developing secondary autoimmunity in MS patient after lymphocyte depletion, compared to MS patients who do not have elevated IL-21.

Identification of a patient by the methods of the invention can be followed by a number of additional steps. For example, the patient may be informed of the increased risk of developing secondary autoimmunity after lymphocyte depletion therapy, or lack of such risk, based on their IL-21 level or genotype. Thus, the invention will allow individualized assessment of the risks of therapy prior to commitment to therapy. The healthcare provider may consider therapeutic options in in view of the risk of secondary autoimmunity, and provide a recommendation, including, for example, administering an IL-21 antagonist before, during, or after lymphocyte depletion therapy, or selecting a treatment regimen that does not involve depletion of lymphocytes.

The healthcare provider may also consider risk management plans for a patient who chooses to undergo lymphocyte depletion therapy. For example, the healthcare provider may inform the patient of the need for further monitoring for the development of secondary autoimmunity after lymphocyte depletion therapy in view of their increased risk of developing secondary autoimmunity. The healthcare provider may also recommend an appropriate monitoring regimen after lymphocyte depletion therapy. An appropriate monitoring regimen for patients at risk may include, without limitation, more frequent monitoring of secondary autoimmunity after lymphocyte depletion therapy at an interval of, for example, one week, two weeks, one month, two months. , three months, six months, or a year. Continued monitoring may be necessary for an extended period of time, for example more than one year, two years, three years, four years, five years or more, because some patients may not present with secondary autoimmunity until long after a period of time. year after lymphocyte depletion therapy. Increased monitoring may also involve, for example, a more comprehensive medical examination (eg, more blood tests) by a specialist to detect any signs of secondary autoimmunity. In addition, pharmacists or clinical staff who distribute a drug that depletes lymphocytes to a patient to treat MS may be required to counsel the patient about the increased risk of developing secondary autoimmunity after use of the drug, in the event that the patient has an elevated IL-21 level and / or has the particular IL-21 genotypes described herein that have been associated with elevated serum IL-21. Pharmacists or clinical staff may also be required to obtain written authorization from the patient before distributing the drug to the patient.

Multiple sclerosis patients

The methods of this invention can be used in the context of any form of MS, for example relapsing-remitting MS, primary progressive MS, and secondary progressive MS. MS patients in the context of this invention are those who have been diagnosed with a form of MS by, for example, symptom history and neurological examination with the help of tests such as magnetic resonance imaging (MRI), punctures spinal cord tests, potential evoked tests, and laboratory analysis of blood samples.

Multiple sclerosis (“MS”), also known as disseminated sclerosis, is an autoimmune condition in which the immune system attacks the central nervous system and leads to demyelination (Compston and Coles, 2008). MS destroys a layer of fat called the myelin sheath that wraps around and electrically insulates nerve fibers. Almost any neurological symptom can appear with the disease, and it often progresses to physical and cognitive disability (Compston and Coles, 2008). MS takes several forms. New symptoms may appear in discrete attacks (recurrent forms) or accumulate slowly over time (progressive forms) (Lublin et al., Neurology 46 (4), 907-11 (1996)). Between attacks, symptoms may disappear completely (remission), but permanent neurological problems often occur, especially as the disease progresses (Lublin et al., 1996). Several subtypes, or patterns of progression, have been described that are important for prognosis and treatment decisions. In 1996, the United States National Multiple Sclerosis Society standardized four definitions of subtype: relapsing-remitting, secondary progressive, primary progressive, and progressive recurrent (Lublin et al., 1996).

The relapsing-remitting subtype is characterized by unpredictable acute attacks, called exacerbations or relapses, followed by periods of months to years of relative calm (remission) without new signs of disease activity. This describes the initial course of most people with MS (Lublin et al., 1996).

Secondary progressive MS begins with a relapsing-remitting course, but subsequently evolves into progressive neurological deterioration between acute attacks without defined periods of remission, although occasional relapses and minor remissions may occur (Lublin et al., 1996).

The primary progressive subtype is characterized by a gradual but steady progression of disability without evident remission after its initial MS symptoms appear (Miller et al., Lancet Neurol 6 (10), 903-12 (2007)). It is characterized by the progression of disability from the beginning, without remissions and improvements, or only occasional and minor (Lublin et al., 1996). The age of onset of the primary progressive subtype is usually later than that of other subtypes (Miller et al., 2007).

Progressive relapsing MS is characterized by a constant neurological decline with acute attacks that may or may not be followed by some recovery. This is the least common of all the subtypes described above (Lublin et al., 1996).

Cases with non-standard behavior have also been described, sometimes referred to as borderline forms of MS (Fontaine, Rev. Neurol. (Paris) 157 (8-9 Pt 2): 929-34 (2001)). These forms include Devic's disease, Balo's concentric sclerosis, Schilder's diffuse sclerosis, and Marburg's multiple sclerosis (Capello et al., Neurol. Sci. 25 Suppl 4: S361-3 (2004); Hainfellner et al., J. Neurol. Neurosurg. Psychiatr 55 (12): 1194-6 (1992)).

Lymphocyte depletion in patients with multiple sclerosis

As used herein, "lymphocyte depletion" is a type of immunosuppression by depletion of circulating lymphocytes, eg, T cells and / or B cells, which results in lymphopenia. Prolonged lymphocyte depletion is seen when, for example, autologous bone marrow transplantation (BMT) or total lymphoid irradiation is used to treat multiple sclerosis. See, for example, Cox et al., Eur. J. Immunol. 35, 3332-3342 (2005). For example, lymphocyte depletion can be achieved through the combined use of thymoglobulin, cyclophosphamide, and whole-body irradiation. Lymphocyte depletion in MS patients can also be achieved through a number of pharmacological treatments. For example, a humanized anti-CD52 monoclonal antibody, CAMPATH-1H (alemtuzumab), has been used in lymphocyte depletion therapy to treat MS patients. CAMPATH-1H-induced lymphopenia has been shown to effectively reduce inflammation of the central nervous system, both clinically and radiologically (Coles et al., Ann. Neurol. 46, 296-304 (1999); Coles et al., 2008) .

Other agents can also be used in lymphocyte depletion therapy to treat MS patients. These agents can be those that cause lymphocyte cell death or inhibit lymphocyte functions. They include, without limitation, (1) agents targeting CD-52-bearing cells, such as agents biologically similar to alemtuzumab, i.e., other anti-CD52 antibodies (eg, chimeric, humanized, or human antibodies) that bind to same epitope or a different epitope such as alemtuzumab or compete with alemtuzumab to bind CD52, and soluble CD52 polypeptides that compete with cell surface CD52 for binding to CD52 ligand (s); (2) biomolecules such as peptides, proteins, and antibodies (eg, chimeric, humanized, or human antibodies) that target cell surface molecules on lymphocytes, such as anti-CD4 antibodies, anti-CD20 antibodies (eg rituximab), anti-TCR antibodies and anti-integrin antibodies (eg natalizumab); (3) cytotoxins (eg, apoptosis inducing agents, cyclophosamide, alkylating agents, and DNA intercalators) delivered specifically or non-specifically to lymphocytes; and (4) antigen-binding portions of the above-mentioned antibodies. Antibodies can include, without limitation, monoclonal antibodies, bifunctional antibodies, oligoclonal antibodies, and polyclonal antibodies.

The term "antigen-binding portion", as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to the same antigen as the full-length antibody from which the portion is derived. Examples of "antigen-binding portion" include, without limitation, a Fab fragment, an F (ab ') 2 fragment, an Fd fragment, a Fv fragment, a dAb fragment, an isolated complementarity determining region (CDR), scFv, and a diabody. Antibodies and antigen-binding portions thereof useful in this invention can be prepared by any method well known in the art.

Any of the above lymphocyte depletion therapies can cause lymphopenia, and in some patients, lymphopenia leads to secondary autoimmunity.

Secondary autoimmunity in MS patients

Autoimmunity is referred to herein as "secondary autoimmunity" when it arises after the onset of a first ("primary") disease, for example a "primary" autoimmune disease. Secondary autoimmunity sometimes arises in MS patients who have or have had lymphopenia after, for example, lymphocyte depletion therapy. In some individuals, secondary autoimmunity arises shortly after lymphocyte depletion therapy (eg, alemtuzumab treatment). In other individuals, secondary autoimmunity may not emerge until months or years after lymphocyte depletion therapy; in some of these individuals, by the time they develop secondary immunity, substantial recovery of lymphocytes (total lymphocyte count) may have occurred so that they are no longer lymphopenic.

Secondary autoimmunity arising in lymphopenic MS patients can be any type of autoimmune condition other than MS, including, but not limited to, thyroid autoimmunity (eg, Graves' disease), immune thrombocytopenia (ITP), Goodpasture's disease , autoimmune neutropenia, autoimmune hemolytic anemia, and autoimmune lymphopenia. Techniques for diagnosing and monitoring these autoimmune diseases are well known to those skilled in the art, including evaluation of symptoms and medical examination, such as blood tests. The invention contemplates the use of any known method. For example, autoantibody levels in a patient's body fluid (eg, blood) can be determined as a means of detecting signs of autoimmunity. Specifically, antinuclear antibodies, anti-smooth muscle antibodies, and anti-mitochondrial antibodies can be measured. In the event that antinuclear antibodies are detected, additional tests can be performed to measure anti-double-stranded DNA antibodies, anti-ribonucleoprotein antibodies, and anti-La antibodies. Anti-thyroid antibodies peroxidase (TPO) and anti-thyroid stimulating hormone (TSH) receptor can be measured to detect autoimmune thyroid diseases; If anti-TPO or anti-TSH receptor antibodies are detected, it can be measured whether thyroid function is affected by measuring the levels of free T3, free T4, and TSH. Antiplatelet antibodies can be measured to detect autoimmune thrombocytopenia; and a measure of blood platelet levels can be used to determine whether the presence of antiplatelet antibodies is causing a reduction in the number of platelets.

IL-21 measurement

In the methods of this invention, IL-21 can be measured by various techniques. IL-21 is a member of the gamma-c family of related cytokines, and has potent activity in promoting T and B cell proliferation and natural killer (NK) cell cytotoxicity. IL-21 is expressed primarily by activated CD4 + T cells (eg, Th17 cells), and is important in type I (Th1) T helper immune responses (Weiss et al., Expert Opin Biol. Ther. 7, 1705 -1721 (2007); Sivakumar et al., Immunology 112, 177-182 (2004)). The human IL-21 gene encodes a polypeptide precursor of 162 amino acid residues and a fully processed mature protein of 133 amino acid residues (about 15 kD); the gene is located on human chromosome 4q26-27 (Sivakumar et al., 2004). The receptor for IL-21 (IL-21 R) has been found in resting peripheral B cells, activated peripheral blood mononuclear cells, and in the germinal center of human lymph nodes (Marleau et al., J. Leukocyte Biol. 78, 575-584 (2005)).

Methods for measuring IL-21 are well known to those of skill in the art. A body fluid sample (eg, blood, serum, plasma, urine, saliva, or cerebrospinal fluid) can be obtained from a patient, and the level of IL-21 in the sample can be measured by any suitable assay for detection. protein, including, but not limited to, immunoassays such as enzyme-linked immunosorbent assays (ELISA). Commercial ELISA kits for measuring human IL-21 are available, for example, from KOMABIOTECH (Seoul, Korea), Bender MedSystems (Burlingame, CA), and eBioscience (San Diego, CA).

Alternatively, the levels of IL-21 transcripts in IL-21 producing cells (eg, Th17 cells) obtained from the patient can be measured by Northern blot analysis and quantitative polymerase chain reaction (Q-PCR). Methods for isolating Th17 cells are well known in the art, and isolation can be done using commercially available kits, for example kits from Miltenyi Biotec (Auburn, CA), eBioscience (San Diego, CA). In some embodiments, IL-21 levels are measured by cytokine staining and flow cytometry in which an anti-IL-21 antibody bound to a detectable moiety is used to detect the intracellular level of IL-21 in cells producing IL-21. IL-21 from the patient. IL-21 can also be measured in terms of activity in a biological assay, for example by measuring proliferative responses of T cells to a combination of IL-21 and IL-15 using, for example, CFSE (carboxyfluorescein succinimidyl ester) (Zeng et al., Curr. Protoc. Immunol. 78: 6.30.1-6.30.8 (2007)). Another method for measuring IL-21 relies on the IL-21-induced tyrosine phosphorylation of Stat3 in splenic CD8 (+) T cells using flow cytometry-based analysis (Zeng et al., 2007). Those skilled in the art will readily appreciate other suitable means of measuring IL-21. In the methods of this invention, the reference value (or index) for determining whether a patient has elevated (abnormally high) IL-21 is the IL-21 value of a control subject, or the mean IL-21 value. from a group of control subjects, obtained using the same test performed at the same time or in a different one. The control subject is a normal or healthy subject, which, in this context, is an individual without any known ongoing inflammatory conditions, including without an autoimmune disease (without any detectable symptoms of an autoimmune disease). In some embodiments, the control subjects are not lymphopenic. An increase in the level of IL-21 by about 10%, 20%, 30%, 40%, 50%, 100%, two times, three times, four times, five times, ten times, twenty times, thirty times, forty times, fifty times, a hundred times, or more can be considered a significant increase. Certain statistical analyzes can be applied to determine if the level of IL-21 in a test sample is significantly different from the control level. Such statistical analyzes are well known to those of skill in the art, and may include, without limitation, parametric (eg, two-tailed Student's t-test) or nonparametric (eg, Wilcoxon's U-test). Mann-Whitney).

Detection of IL-21 SNP genotypes

In some methods of this invention, genotyping is used to predict whether a patient is prone to have (i.e., at risk of having or likely to have) elevated IL-21 and thus at risk of developing secondary autoimmunity while having lymphopenia. "Genotyping" refers to the process of determining the genotype of an individual through the use of biological tests. Genotyping methods are well known to those of skill in the art, and include, without limitation, PCR, DNA sequencing, allele-specific oligo (ASO) probes, and hybridization with DNA microarrays or beads. Genotyping can be partial, that is, only a small fraction of an individual's genotype is determined. In the context of this invention, only certain SNPs need to be detected. A SNP is a DNA sequence variation that occurs when a single nucleotide - A, T, C, or G - in a corresponding portion of the genome differs between members of a species or between paired chromosomes in an individual. Known human SNPs are assigned SNP identification numbers from reference (refSNP or rs) in the Single Nucleotide Polymorphism Database (dbSNP) public domain file housed at the National Center for Biotechnology Information (NCBI).

The present inventors have discovered that the minor SNP genotypes of rs13151961 A / A, rs6822844 G / G and rs6840978 C / C are associated with significantly higher levels of IL-21 in serum compared to people who do not have these genotypes. MS patients who have one or more of these SNP phenotypes thus have a greater susceptibility to developing secondary autoimmunity after lymphocyte depletion, compared to MS patients who do not have these genotypes.

Timing of obtaining information about IL-21

Obtaining information on IL-21 (IL-21 levels or IL-21 related SNP genotypes) from a patient with MS is helpful in selecting treatment and post-treatment monitoring regimens for the patient. When the information is obtained prior to MS therapy, the patient can be informed about the relative risk of developing secondary autoimmunity after lymphocyte depletion therapy, and treatment decisions can be made accordingly. The patient may also be informed of the need for further post-treatment monitoring, for example a more frequent and thorough examination by a specialist, if classified as "at risk". In this way, IL-21 information improves risk management (by physicians, pharmacists and patients) in the treatment of MS.

Obtaining IL-21 information during or after MS treatment will also be useful for monitoring the development and treatment of secondary autoimmunity. As noted above and described below, we have found that after lymphocyte depletion therapy, MS patients who develop secondary autoimmunity have a much greater increase in their serum IL-21, compared to MS patients who they do not develop secondary autoimmunity. The latter group of MS patients produces only slightly more IL-21 after lymphocyte depletion. Thus, by measuring IL-21 production after lymphocyte depletion treatment, the risk of secondary autoimmunity can also be predicted, which may not occur until months or years after treatment.

Treatment of secondary autoimmunity

A secondary autoimmune disease that arises in patients with MS can be treated depending on the type of disease. Secondary autoimmunity can be treated using an effective dose of an IL-21 antagonist. An IL-21 antagonist can be a therapeutic agent that inhibits the activity of IL-21, for example an agent that inhibits the interaction between IL-21 and IL-21 R. "An effective dose" refers to the amount of a inhibitory agent sufficient to inhibit IL-21 activity in a patient in such a way as to alleviate or prevent the symptoms of secondary autoimmune disease. IL-21 antagonists can be, for example, chimeric, humanized or human monoclonal antibodies against human IL-21 or IL-21R proteins, or soluble IL-21R. See also, for example, U.S. Patent No. 7,410,780 and U.S. Patent Application Publication No. No. 20080241098. Pharmaceutical compositions containing an IL-21 antagonist can be prepared according to methods known to those skilled in the art. Pharmaceutical compositions containing IL-21 antagonists can be administered to a patient using a suitable method known in the art, for example intravenously, intramuscularly or subcutaneously.

Kits to treat and evaluate patients with MS

The present description provides kits for treating multiple sclerosis. A kit may contain, among others, a drug that depletes lymphocytes (for example, alemtuzumab) and a written instruction to inform a patient or healthcare provider of the drug's contraindications, for example, the possibility of increased risk of developing a secondary autoimmune disease after treatment with the drug. The increased risk may be associated with or indicated by (i) elevated IL-21, or (ii) the presence of one or more genotypes of single nucleotide polymorphisms (SNPs) selected from the group consisting of: A / A in SNPs rs13151961, G / G in SNP rs6822844, and C / C in SNP rs6840978.

The disclosure also provides kits for detecting IL-21 in serum in a MS patient, and / or for identifying MS patients at increased risk of developing secondary autoimmune disease after lymphocyte depletion. Such kits may comprise an anti-IL-21 antibody, or an antigen-binding portion thereof, or a soluble IL-21 receptor, and optionally an instruction instructing the user to take a blood sample from a patient, and optionally one or more reagents to detect binding of IL-21 antibody, portion or soluble receptor to IL-21 in the blood sample of the MS patient. Such kits will have been validated or approved by an appropriate regulatory authority to perform medical diagnoses in patients, such as patients with MS.

The disclosure also provides kits for identifying an MS patient who is at increased risk of developing a secondary autoimmune disease after lymphocyte depletion. The kits may comprise one or more reagents suitable for identifying the presence or absence of one or more genotypes of SNPs selected from the group consisting of: SNP rs13151961, SNP rs6822844, and SNP rs6840978, in a sample obtained from a patient with MS, and an instruction that directs a user to take a sample from a patient with MS.

Assessment of T-cell responsiveness to MS treatment

This disclosure provides methods for evaluating the responsiveness of T cells to lymphocyte depletion therapy treatment in a patient with MS. The methods involve measuring caspase-3 on T cells obtained from the patient after treatment. An increase in caspase-3 (eg, caspase-3 protein, RNA transcript, and / or activity levels) in T cells compared to T cells from a non-treated MS patient indicates that the cells T in the treated patient have responded to treatment. These methods are based on our discovery that T cells from people with untreated MS are resistant to apoptosis, and that this resistance is associated with under-expression of caspase-3. But after lymphocyte depletion therapy, caspase-3 expression increases significantly in T cells, reaching levels seen in healthy people.

Techniques for measuring caspase-3 in T cells are well known in the art. For example, one can obtain T-cell cell extracts using techniques well known in the art, and measure caspase-3 protein levels by, for example, ELISA. Commercial ELISA kits for measuring human caspase-3 are available from, for example, Bender MedSystems (Burlingame, CA), EMD Chemicals, Inc. (San Diego, CA), and R&D Systems, Inc. (Minneapolis, MN). . Alternatively, caspase-3 transcript levels can be measured in T cells by, for example, Northern blot analysis or quantitative PCR. Caspase-3 can also be measured in terms of activity in a biological assay, for example by measuring its protease activity. Commercial kits for measuring caspase-3 activity are available from, for example, Roche Applied Science (Indianapolis, IN), and Invitrogen (Carlsbad, CA).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or evaluation of the present invention. In case of conflict, this specification, including definitions, will prevail. Although various documents are cited herein, this quotation does not constitute an admission that any of these documents are common general knowledge in the art. Throughout this specification and embodiments, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of an integer or group of integers but not the exclusion of any other number. whole or group of whole numbers. The materials, methods, and examples are illustrative only and are not intended to be limiting. The following examples are intended to illustrate the methods and materials of the present invention.

EXAMPLES

In the following examples, all patients had relapsing-remitting multiple sclerosis and participated in one of two clinical trials: CA ^m M ^s -223 and CAMMS-224 (REC 02/315 and 03/078) in which alemtuzumab was administered. by intravenous infusion of 12-24 mg / day for five days, followed by a new treatment at 12 months. Patients and controls accepted research phlebotomy (LREC 02/263), and all were free from exposure to other disease-modifying agents, including steroids, for at least one month at the time of blood sampling.

Lymphocyte proliferation and apoptosis data were generated by a cross-sectional ex ^vivo fresh cell study of 65 patients and 21 healthy controls (7 males, mean age 34 years). This generated hypotheses about T cell cycling in the pathogenesis of secondary autoimmunity, which were tested in samples available nine months after alemtuzumab treatment, which was chosen as the earliest time point at which cell apoptosis T could be robustly analyzed. Of the 29 samples available at this time, 10 met our study's definition of autoimmunity (1 man, mean age 36 years) compared with 10 without autoimmunity (3 men, mean age 38 years). Autoimmunity was defined as the development of a new autoimmune disease (with or without autoantibodies), or persistent significant titers of autoantibodies (present on at least two occasions at least three months apart) without clinical disease. "No autoimmunity" was defined as the absence of an autoimmune disease and autoantibodies for at least 18 months after alemtuzumab in this study. Of the ten patients with autoimmunity, three had autoantibodies only (antinuclear antibodies). Serum IL-21 was then measured serially in: 15 randomly selected patients with autoimmunity - five of whom had been studied as previously (three men, mean age 34 years; twelve with thyroid autoimmunity, one with thyroid disease). Goodpasture, one with ITP, and one with antinuclear antibodies only), and fifteen randomly selected patients without autoimmunity - six of whom had been studied as previously (five men, mean age 31 years) and nineteen healthy controls (seven men, age mean 33 years).

73 subjects were studied for genetic analysis. Of these, 23 met the definition of “no autoimmunity,” and 27 had secondary autoimmunity after alemtuzumab (six with autoantibodies only: four with antinuclear antibodies and two with anti-smooth muscle antibodies; eighteen with thyroid autoimmunity, two with ITP and one with Goodpasture's disease). The remaining 23 subjects could not be classified according to transient autoantibody production and / or insufficient time since alemtuzumab treatment.

For all statistical analyzes described in the following examples, the data was analyzed using SPSS 12.0.1 for Windows. After evaluation of normality, parametric (Student's t test) or non-parametric (Wilcoxon-Mann-Whitney) tests were performed. P values are expressed throughout the text, in which a value of p <0.05 was considered statistically significant, modified by a Bonferroni correction when indicated.

Example 1: Alemtuzumab induces T-cell lymphopenia.

A single dose of alemtuzumab resulted in depletion of CD4 + and CD8 + T cells at 5.6% and 6.8% respectively of baseline values at month 1, and 30.3% and 40.8% respectively at month 1. month 12 (data not shown).

Example 2: T cells from untreated multiple sclerosis patients are resistant to cell death For various tests performed in this Example, different cross-sectional and longitudinal samples were used according to availability. As a prelude to measuring the cell cycle of lymphocytes after alemtuzumab, we examined the proliferative response of T cells, unstimulated or in culture with myelin basic protein (MBP) or thyroid stimulating hormone receptor (TSHr), between untreated patients with multiple sclerosis and normal controls (FIGS. 1A and 1B).

A. Peripheral mononuclear cell cultures

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by centrifugation on a Ficoll-Paque density gradient (Amersham Pharmacia Biotech). Whole PBMCs were immediately suspended in culture medium (RPMI) containing 1% penicillin, 1% streptomycin and 10% fetal calf serum (Sigma S5394), and adjusted to a concentration of 106 / ml viable cells. (determined by exclusion with trypan blue). To induce passive cell death, PBMCs were incubated for 72 hours in media alone, without additional growth factors. Fas-mediated apoptosis was induced by culturing PBMCs for 48 hours with soluble anti-CD28 (1 mg / ml: kindly donated by M. Frewin, University of Oxford) in plates pre-coated with anti-CD3 mAb (1 mg / ml - BD Pharmingen), followed by an 18 hour incubation with activating anti-human Fas (clone CH11, 1 mg / ml - Upstate Biotechnology, Lake Placid, NY).

B. Detection of apoptosis

Apoptotic T cells were detected by cell staining with: allophycocyanin-conjugated mouse anti-human monoclonal antibodies against CD3 (Serotec MCA463APC), CD4 (Serotec MCA1267APC) and CD8 (Serotec MCA1226APC), annexin V conjugated with FITC and propidium iodide Pharmingen). Fluorescence was detected by flow cytometry (FACSCALIBUR: Becton Dickinson, Mountain View, CA). Based on the forward and side scatter, a broad lymphocyte gate was drawn to include both live and apoptotic lymphocytes (having reduced FSc and increased SSc). At least 15,000 in-gate events were collected and analyzed using WinMDI 2.8 software. Early apoptotic cells were defined as annexinV + PI-, and late apoptotic or necrotic cells as annexinV + PI + (Aubry et al., Cytometry 37, 197-204 (1999)). Apoptotic cell death was defined as total cell death (annexinV + PI- plus annexinV + PI +) blocked by inhibition of pancaspase with Q-VD-OPh (RnD Systems OPH001).

C. Proliferation assays

PBMCs were loaded with the cell division monitoring dye CFSE (carboxyfluorescein diacetate succinimidyl ester) (Lyons et al. Methods Cell Biol. 63, 375-398 (2001)), and cultured with 50 mg / ml protein basic myelin (MBP: RDI-TRK8M79 / LYO) or 1 mg / ml extracellular domain of thyroid stimulating hormone receptor bound to a matrix-binding protein (TSHr: kindly donated by M. Ludgate, University of Cardiff). After 10 days, CFSE staining in cells, identified by specific surface markers (CD4, CD8), was analyzed by flow cytometry. The precursor frequencies (defined as the proportion of lymphocytes that allowed the parental population to undergo at least two cell divisions) and the proliferation index (defined as the sum of the cells in all generations divided by the calculated number of parental cells) are calculated using Modfit LT 3.0 (Verity Software). The absolute number of surviving cells was measured in comparison with a fixed number of inert beads (BD CALIBRITE, BD Biosciences), embedded in cultures.

D. Results

There were no differences in the proliferative response of T cells, unstimulated or in culture with myelin basic protein (MBP) or the thyroid stimulating hormone receptor (TSHr), between untreated patients with multiple sclerosis and normal controls ( FIGS. 1A and 1B). In contrast, T-cell survival from untreated multiple sclerosis patients was> 4 times that of controls (p <0.005; FIG. 1C), suggesting that reduced T-cell death is a characteristic of untreated multiple sclerosis. We confirmed this by demonstrating that T cells from untreated patients are resistant to both passive and Fas-mediated apoptosis compared to healthy controls (passive: 0.3% vs. 6.7%, p = 0.0016; and mediated by Fas: 2.9% vs 15.5%, p = 0.0018; FIGS. 1E and 1F).

Example 3: T cells that regenerate after alemtuzumab are highly proliferative, prone to self-reactivity, and susceptible to apoptosis

Using the assays described in Example 2, we found that after alemtuzumab, the proportion of T cells responding to autoantigens (precursor frequencies) and the degree of proliferation (proliferative index) were significantly increased compared to untreated patients and controls. healthy. For example, at month 3, unstimulated T cell proliferation was> 6.5 times that of untreated patients, and proliferation in response to stimulation with MBP and r TSH increased by 900% and 700% respectively ( all p <0.01: FIGS. 1A and 1B). T cell apoptosis was also significantly increased after alemtuzumab. In response to antigenic stimulation, the proportion of T cells that underwent apoptosis at six months was 10 times higher than at baseline (FIG. 1D; p <0.001 for all antigens), resulting in fewer viable T cells at baseline. end of culture (FIG. 1C). Passive and Fas-mediated apoptosis also increased after alemtuzumab, with rates at least twice those seen in the healthy control group (passive: 24.5%, 22.2%, and 17.9% at 6, 9 and 12 months, respectively, compared to 6.7% in controls, all p <0.001; Fas-mediated 37.8%, 35.8%, and 29.9% at 6, 9, and 12 months, respectively, in compared with 15.5% in controls, all p <0.01; FIGS. 1E and 1F). An increase in lymphocyte apoptosis was observed after alemtuzumab in the CD4 + and CD8 + subpopulations (FIGS. 1G and 1H), and persisted for at least 18 months after treatment with alemtuzumab (data not shown).

Example 4: T cells from untreated multiple sclerosis patients underexpress caspase 3

A. mRNA analysis

PBMCs, immediately ex ^vivo or after culturing with MBP or polyclonal stimulation, were positively separated, using 20 µl of magnetic beads (Miltenyi Biotec; CD19 Microbeads, CD3 Microbeads, CD14 Microbeads) per 1x107 cells loaded on a MACS® LS column. Magnetically retained cells were eluted, washed and stored in RNA / afer ™ at -70 ° C (consistent cell purity 95-98%, data not shown).

The expression of Fas, FasL, Bcl-2, Bcl-Xl, Bad, Bax, Bid, Bim, Survivin, c-FLIP, and Caspase 3, 8 and 9 was determined by semi-quantitative RT-PCR. The mRNA was extracted from cells stored in RNA / afer ™ using the RNEASY Mini Kit (QIAgen), and reverse transcribed to cDNA using the PRO-STAR First Strand RT-PCR Kit (Stratagene). PCR primers and probes were designed using PRIMER EXPRESS (PE Biosystems, Foster City, CA, USA), and were purchased from Oswel DNA service. The mRNA sequence information was obtained from GenBank. Quantitative real-time PCR was performed on an ABI Prism 7900HT sequence detection system (Perkin Elmer) using ROX-containing PCR Mastermix (Eurogentec RT-QP2X-03). The sequences of the primers and probes were: Bcl-2 for: 5'-CCT GTG GAT GAC TGA GTA CCT GAA-3 '(SEQ ID NO: 1), Rev 5'-CAC CTA CCC AGC CTC CGT TA-3 '(SEQ ID NO: 2), JOE labeled probe 5'-CGG CAC CTG CAC ACC TGG ATC-3' (SEQ ID NO: 3); Bcl-X1 for 5'-TTC AGT CGG AAA TGA CCA GAC A-3 '(SEQ ID NO: 4), Rev 5'- GAG GAT GTG GTG GAG CAG AGA-3' (SEQ ID NO: 5), labeled probe with FAM 5'-TGA CCA TCC ACT CTA CCC TCC CAC CC-3 '(SEQ ID NO: 6); Fas for 5'- AAA AGC ATT TTG AGC AGG AGA GTA TT-3 '(SEQ ID NO: 7), Rev 5'- GGC CAT TAA GAT GAG CAC CAA-3' (SEQ ID NO: 8), probe labeled with JOE 5'- CTA GAG CTC TGC CAC CTC TCC ATT -3 '(SEQ ID NO: 9); FasL for 5'- AAG AAA GTG GCC CAT TTA ACA G-3 '(SEQ ID NO: 10), Rev 5'- AGA AAG CAG GAC AAT TCC ATA GGT-3' (SEQ ID NO: 11), probe labeled with FAM 5'- CAA CTC AAG GTC CAT GCC TCT GG-3 '(SEQ ID NO: 12); Survivin for 5'- CTG CCT GGC AGC CCT TT-3 '(SEQ ID NO: 13), Rev 5'- CTC CAA GAA GGG CCA GTT CTT - 3' (SEQ ID NO: 14), 5 'FAM labeled probe - TCA AGG ACC ACC GCA TCT CTA CAT T-3 '(SEQ ID NO: 15); c-FLIP for 5'- GTG GAG ACC CAC CTG CTC -3 '(SEQ ID NO: 16), Rev 5'- GGA CAC ATC AGA TTT ATC CAA ATC C -3' (SEQ ID NO: 17), labeled probe with FAM 5'-CTG CCA TCA GCA CTC TAT AGT CCG AAA CAA -3 '(SEQ ID NO: 18); Caspase 8 for 5'- AGG AGG AGA TGG AAA GGG AAC TT -3 '(SEQ ID NO: 19), Rev 5'- ACC TCA ATT CTG ATC TGC TCA CTT CT -3' (SEQ ID NO: 20), probe labeled with JOE 5'-CTC CCT ACA GGG TCA TGC TCT ATC AGA TTT CAG -3 '(SEQ ID NO: 21); Caspase 3 for 5'- AAG ATC ATA CAT GGA AGC GAA TCA -3 '(SEQ ID NO: 22), Rev 5'- CGA GAT GTC ATT CCA GTG CTT TTA -3' (SEQ ID NO: 23), labeled probe with FAM 5'-CTG GAA tAt CCC TGG ACA ACA GTT ATA AA -3 '(SEQ ID NO: 24); Caspase 9 for 5'-TGC GAA CTA ACA GGC AAG CA -3 '(SEQ ID NO: 25), Rev 5'- GAA CCT CTG GTT TGC GAA TCT C -3' (SEQ ID NO: 26), probe labeled with FAM 5'- CAA AGT TGT CGA AGC CAA CCC TAG AAA ACC TTA -3 '(SEQ ID NO: 27); Bad for 5'- CAG TGA CCT TCG CTC CAC ATC - 3 '(SEQ ID NO: 28), Rev 5' - ACG GAT CCT CTT TTT GCA TAG -3 '(SEQ ID NO: 29), JOE 5 labeled probe '- ACT CCA CCC GTT CCC ACT GCC C-3' (SEQ ID NO: 30); Bax for 5'-TTT CTG ACG GCA ACT TCA ACT -3 '(SEQ ID NO: 31), Rev 5'-GGT GCA CAG GGC CTT GAG-3' (SEQ ID NO: 32), JOE labeled probe 5'- TGT CGC CCT TTT CTA CTT TGC CAG CA-3 '(SEQ ID NO: 33); Bid for 5'-GCT GTA TAG CTG CTT CCA GTG TAG -3 '(SEQ ID NO: 34), Rev 5'-GCT ATC TTC CAG CCT GTC TTC TCT -3' (SEQ ID NO: 35), probe labeled with JOE 5'-AGC CCT GGC ATG TCA ACA GCG TTC -3 '(SEQ ID NO: 36) and Bim for 5'-ACC ACA AGG ATT TCT CAT GAT ACC -3' (SEQ ID NO: 37), Rev 5 ' - CCA TAT GAC AAA ATG CTC AAG GAA -3 '(SEQ ID NO: 38), probe labeled with FAM 5'- TAG CCA CAG CCA CCT CTC TCC CT-3' (SEQ ID NO: 39). B. Results

T-cell mRNA expression of caspase 3, the effector caspase common to both apoptotic pathways, from untreated multiple sclerosis patients was reduced compared to controls; this was significant for unstimulated and MBP-stimulated PBMCs (in 78% and 87% respectively, both p <0.05, after correction for multiple comparisons), but not for stimulated polyclonal cultures (FIG. 2A). A similar trend was observed in CD14 + cells (but not in CD19 + cells), although this difference did not survive correction for multiple comparisons (FIG. 2B). After alemtuzumab, the expression of caspase 3 increased significantly in T cells and monocytes, reaching levels observed in healthy controls (p <0.05; FIGS. 2A and 2B). The expression of all other genes tested (listed in the methods) did not change after alemtuzumab.

Thus, our studies show that T cells from people with untreated multiple sclerosis are resistant to apoptosis, and this resistance is associated with under-expression of caspase 3. According to the position of this effector caspase at the point of convergence From extrinsic and intrinsic apoptotic pathways, we have demonstrated resistance of T cells to both passive and Fas-mediated apoptosis in our patients. Caspase 3 under-expression has been described in some autoimmune diseases, including type I diabetes (Vendrame et al., Eur J Endocrinol 152, 119-125 (2005)), Hashimoto's thyroiditis, and autoimmune poliendocrine syndrome 2 (Vendrame et al. , J Clin Endocrinol Metabjc (2006)). However, this is a novel finding in multiple sclerosis.

Example 5: Secondary autoimmunity after alemtuzumab is associated with excessive T-cell apoptosis

After having demonstrated increased lymphocyte proliferation and apoptosis as a generic response to treatment, we tested the relationship between T cell apoptosis and the development of autoimmunity after alemtuzumab, defined as the development of a new autoimmune disease and / or persistent autoantibodies by above normal range, after alemtuzumab, sustained for at least 3 months. Using this definition, T cells derived from autoimmune patients (n = 10) showed significantly higher levels of apoptotic cell death in all culture conditions at 9 months after treatment, compared to T cells from non-autoimmune patients. (n = 10) studied at the same time point (unstimulated 4.7% vs. 14.4%, mediated by Fas 18.2% vs. 32.1%, MBP 7.6% vs. 17.6 %, and TSHr 9.5% vs 25.5%, p <0.01 for all comparisons; FIG. 3). If a stricter definition of autoimmunity was applied, defined as the development of an autoimmune disease, excluding the production of non-pathogenic antibodies, the difference was maintained, despite reducing the number in the autoimmune group to 7 (not stimulated, 4, 7% vs. 15.4%, Fas-mediated, 18.2% vs. 31.7%, MBP, 7.6% vs. 20.2%, TSHr, 9.5% vs. 13.4%; P <0.02 for all comparisons).

There was no difference in the rate of T cell reconstitution between the two groups (for example, at 6 months, CD4 counts are 0.15 x 109 / L vs 0.19 x 109 / L; and CD8 counts are 0.11 x 109 / l vs 0.11 x 109 / l in those with and without autoimmunity, respectively), suggesting a higher T cell cycle in the autoimmune group (data not shown).

Example 6: IL-21 induces T cell proliferation and apoptosis

A. IL-21 Assays and Enrichment

Serum IL-21 was measured using the EBIOSCIENCE kit (88-7216-86) according to instructions. Plates were read using a microplate reader (model 680, BioRad) at 450 nm. Polyclonally stimulated and unstimulated PBMCs (1 mg / ml plate-bound anti-CD3 and 1 mg / ml soluble anti-CD28) were added with 5 pg / ml and 20 pg / ml rhIL-21 (EBIOSCIENCE 14- 8219). Apoptosis and proliferation of CD4 + and CD8 + were assessed as described above.

B. Results

We evaluate the effect of exogenous IL-21 on apoptosis and proliferation of human T cells in vitro. Addition of PBMCs from healthy rhIL-21 controls led to an increase in apoptotic death of unstimulated and polyclonally stimulated CD4 + (FIG. 4A) and CD8 + (FIG. 4B) T cells, in a dose-dependent manner ( p <0.05 for all conditions). The addition of cells not stimulated with rhIL-21 led to a small but significant increase in the proliferation of CD4 + and CD8 + T cells; with an increase both in the index proliferative (CD4 + and CD8 +: 1.07 vs 1.25, p = 0.017; and 1.09 vs 1.32, p = 0.017, respectively; FIG. 5A) and precursor frequency (CD4 + 0.007 vs 0.014, p = 0.016; CD8 + 0.007 vs 0.015, p = 0.026; FIG. 5C). IL-21 did not affect the proportion of CD4 + or CD8 + T cells that proliferate in response to polyclonal stimulation (FIG.

5D), suggesting that they were already maximally stimulated. IL-21, however, led to a significant increase in the degree of proliferation of CD8 + cells (proliferative index 11.59 vs. 19.39, p = 0.012; FIG. 5B).

Example 7: IL-21 predicts the development of secondary autoimmunity after alemtuzumab

At all time points in Example 6, the serum IL-21 concentration was significantly higher in patients who developed secondary autoimmunity compared to the non-autoimmune group (for all comparisons p <0.05; FIG. 6A). We examined all pretreatment serum samples from 84 patients who subsequently received alemtuzumab. After at least two years of follow-up with these patients, we categorized them as belonging to the “autoimmune” group (n = 35: 32 patients with thyroid diseases, one with ITP, one with Goodpasture's disease, and one with alopecia) or “non-autoimmune” group (n = 49: patients without or with only transient autoantibodies (not sustained for six months)). These pretreatment sera had a statistically higher mean concentration of IL-21 than the controls; however, this was explained by the high levels of IL-21 in those patients who developed secondary autoimmunity. There was a highly significant difference in pretreatment mean IL-21 levels between those who developed autoimmunity (464 pg / ml) and those who did not (229 pg / ml; p = 0.0002) (FIG. 6B).

The association between induced lymphopenia and autoimmunity has been observed in animal models. Under lymphopenic conditions, the remaining T cells undergo extensive compensatory expansion to reconstitute the immune system. This process, called homeostatic proliferation, is based on stimulation through the TCR-auto-peptide-MHC complex (Ge et al., PNAS 101, 3041-3046 (2004); Ge et al., PNAS 98, 1728-1733 (2001); Kassiotis et al., J Exp. Med. 197, 1007-1016 (2003)) and results in a population prone to increased recognition of the autoantigen, as observed in our studies. In addition, rapidly expanding T cells acquire the phenotype and functional characteristics of memory cells, including: less dependence on costimulation, the ability to respond to lower doses of antigen than naive cells, and rapid secretion. of inflammatory cytokines in restimulation, thus promoting the breakdown of self-tolerance (Cho et al., J Exp. Med. 192, 549-556 (2000); Goldrath et al. J Exp. Med. 192, 557-564 (2000); Murali-Krishna et al., J Immunol 165, 1733-1737 (2000); Wu et al., Nat Med. 10, 87-92 (2004)). However, despite these changes, autoimmunity is not an inevitable consequence of lymphopenia. In fact, as with our patients, the majority of lymphopenic subjects did not develop autoimmunity, suggesting that additional "cofactors" are required.

We have demonstrated here for the first time in man that IL-21 overproduction is the required "second target" in the development of secondary autoimmunity after the otherwise successful treatment of multiple sclerosis with a lymphocyte depletion agent such as alemtuzumab. Our studies show that autoimmunity arises in patients with lymphocyte depletion, with increased T-cell apoptosis, and a cell cycle driven by genetically influenced higher levels of IL-21 that are detectable even before treatment. Even before treatment, patients who developed secondary autoimmunity had serum IL-21 levels more than twice that of the non-autoimmune group, suggesting that serum IL-21 may serve as a biomarker for the risk of developing autoimmunity months to months. years after alemtuzumab treatment. Without wishing to be bound by theory, we believe that, by driving T cell expansion and death cycles to excess, IL-21 increases the likelihood of generating autoreactive T cells and thus autoimmunity. Thus, cytokine-induced abnormal T-cell cycling is a general principle of lymphopenia-associated autoimmunity.

Example 8: IL-21 genotype influences IL-21 expression and is associated with autoimmunity

A. Genotyping of IL-21

In total, four SNPs, rs13151961, rs6822844, rs4833837, and rs6840978, found in a tightly bound disequilibrium region containing four genes, KIAA1109-ADAD1-IL2-IL21, on chromosome 4q27 were tested. All four SNPs were available as Assay-On-Demand (AoD) products from Applied Biosystems. The genotyping of the SNPs was carried out using the TaqMan methodology of Applied Biosystems according to the conditions recommended by the manufacturer. Polymerase chain reaction (PCR) was performed on Applied Biosystems 384-well 9700 Viper PCR machines, after which genotypes were localized on a 7900 high-throughput sequence detection system (SDS) using the software. SDS Version 2.1. Each individual was genotyped in duplicate. All individuals were further genotyped for the genetic factors associated with multiple sclerosis: HLA-DRB1 * 1501, rs2104286 (L2RA) and rs6897932 (IL7R).

B. Results

To determine if there is an association between genetic variation and IL-21 production, we genotyped 73 subjects, in whom the pre-alemtuzumab serum IL-21 concentration was determined, for four polymorphisms of

Claims (12)

1. A method of selecting a multiple sclerosis (MS) patient for lymphocyte depletion therapy, comprising the steps of: a) measure the level of IL-21 in a patient's blood sample, wherein the blood sample is obtained prior to any lymphocyte depletion therapy, b) comparing the level of IL-21 with the level of IL-21 in a control blood sample from a subject without an autoimmune disease, and c) selecting the patient for lymphocyte depletion therapy if the level of IL-21 in the patient's blood sample is not elevated compared to the level of IL-21 in the control blood sample. The method of claim 1, wherein the measure is of mRNA encoding IL-21 in IL-21 producing cells in the sample. 3. The method of claim 2, wherein the IL-21 producing cells are Th17 cells. 4. The method of claim 1, wherein the measure is intracellular IL-21. The method of claim 4, wherein the measurement comprises staining and flow cytometry. 6. The method of claim 1, wherein the measurement is serum IL-21. The method of claim 6, wherein the measurement comprises the use of an enzyme-linked immunosorbent assay (ELISA). The method of any one of claims 1-7, wherein the lymphocyte depletion therapy comprises administering an anti-CD52 antibody to the patient. 9. The method of claim 8, wherein the anti-CD52 antibody is alemtuzumab. 10. A method of selecting a multiple sclerosis (MS) patient for lymphocyte depletion therapy, comprising the steps of: a) genotype a patient's DNA sample to detect the presence or absence of one or more genotypes of single nucleotide polymorphisms (SNPs) selected from the group consisting of: A / A in SNP rs13151961, G / G in SNP rs6822844, and c / c in SNP rs6840978, in which the DNA sample is obtained prior to any lymphocyte depletion therapy, and b) selecting the patient for lymphocyte depletion therapy if said genotypes of SNPs are not detected in the DNA sample. The method of claim 10, wherein the lymphocyte depletion therapy comprises administering an anti-CD52 antibody to the patient. 12. The method of claim 11, wherein the anti-CD52 antibody is alemtuzumab.